(1) Field of the Invention
The field of this invention is nucleic acid chemistry, more specifically the field that covers methods for increasing the number of DNA molecules that have a preselected target sequence (“amplifying” that target sequence), and most specifically the field that covers amplification procedures that are done isothermally, without the temperature cycling used in the classical polymerase chain reaction.
(2) Description of Related Art
For practical applications in many areas, including diagnostic procedures that target DNA- and RNA-molecules in biological samples, methods are desired that “amplify” specific nucleic acid sequences. Classically, this has been done by the polymerase chain reaction (PCR) [R. K. Saiki, D. H. Gelfand, S. Stoffel, S. J. Scharf, R. Higuchi, G. T. Horn, K. B. Mullis, H. A. Erlich (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487-491]. Here, a “forward primer” that binds to a pre-selected target oligonucleotide is annealed to the target sequence to form a duplex. Then, the primer is incubated with a DNA polymerase and the appropriate 2′-deoxynucleoside triphosphates to yield a product that is complementary (in the Watson-Crick sense) to the target oligonucleotides; the target and its complement, as it is formed, are bound in a double stranded double helix. The double strand is then “melted” by heating, typically to temperatures above 75° C., yielding the two complementary DNA strands as single strands. Each strand is freed by heating from its complement. The original target is then able to bind to a second forward primer, while the product DNA molecule is able to bind to a “reverse primer”, which is designed to bind to a preselected site downstream in the product DNA molecule. The polymerase extension is then repeated, with both primers extended to give full-length products, again as duplexes (now two in number). The two strands are then separated by heating to allow more forward and reverse primer to anneal, and the cycle is repeated. The results are multiple copies of both the target DNA molecule and its complement. In asymmetric PCR, the ratio of these two is different from unity.
Classical PCR is widely used throughout research, science, and technology, being the method of choice to detect small amounts of DNA in complex biological samples. Nevertheless, the use of temperature cycling to separate the two strands in product duplexes is undesirable in many applications, including applications that want to amplify target DNA at points-of-care, in doctors offices, and in the field. The desire to amplify target DNA molecules without needing to do repeated temperature cycling is indicated by the literature that searches for amplification methods that do not need temperature cycling, including those known as “recombinase polymerase amplification” (RPA) [Piepenburg, O., Williams, C. H., Stemple, D. L., Armes, N. A. (2006) DNA Detection using recombination proteins. PLoS Biol 4 (7): e204], rolling circle amplification (RCA), NASBA, helicase-dependent amplification (HDA) [Tong, Y., Lemieux, B.,; Kong, H. (2011) Multiple strategies to improve sensitivity, speed and robustness of isothermal nucleic acid amplification for rapid pathogen detection. BMC Biotechnol. 11 Art. No: 50] [Lemieux, B., Li, Y.; Kong, H. M., Tang, Y. W. (2012) Near instrument-free, simple molecular device for rapid detection of herpes simplex viruses: Expert Review Molec. Diagnostics 12, 437-443 DOI: 10.1586/ERM.12.34] and LAMP, among others. These are called “isothermal amplification” methods.
Isothermal amplification methods frequently do not perform well, however. In many cases, the extent of amplification appears to depend on the specific sequence being amplified or (perhaps) the sequence of probes and/or primers used in the amplification. In some cases, the amplification fails entirely. In many cases, extra “spurious” products are observed to arise in addition to the target amplicon. Spurious products are especially seen when isothermal amplification is attempted for more than one target nucleic acid in a single sample (“multiplexing”).
Essentially no theory explains these and other variable results, although speculation can be found in the public and private art, some of it contradictory, other explanations being informal. Without any attempt to be exhaustive, speculative suggestions include the possibility that at low temperatures, non-Watson Crick interactions might cause some of the DNA molecules involved (primer, probe, or analyte) to fold in a way that defeats the amplification process. Others have suggested that high temperatures must be regularly traversed to avoid an (often unknown) intra- or intermolecular interaction from capturing the system as an artifact. Primer-primer interactions have been invoked to explain failure of various isothermal amplification systems, especially when is multiplexing is attempted.
None of these explanations are established. Few data allow us to prefer one over another. As a consequence, the art contains no clear guidance as to what experiments might be tried to overcome these problems, and to generate reliable procedures of performing isothermal amplification for all target sequences and, especially, for multiple (more than one) target sequences.
This is especially true for the isothermal amplification method known as helicase-dependent amplification (HDA). Instead of raising the temperature to separate product duplexes, HDA uses a protein known as helicase. In theory, helicase pulls two strands apart to allow primers to bind to create two duplexes from an original single duplex. While HDA creates successful amplification for many targets, it unfortunately does not for most targets. Again, additional products are often seen with HDA targeting single DNA molecules, often causing the isothermal amplification to fail. Attempts to multiple HDA nearly always fail. Again, while the spurious products are occasionally called “primer dimers”, few if any examples exist where those structures are proven. In any case, formation of these spurious products limits sensitivity and multiplexing. Further, standard HDA cannot use primer concentrations higher than ca. 0.2 μM which limit the speed of detection.